CD14 is not required for carbon tetrachloride-induced hepatic inflammation and fibrosis with or without lipopolysaccharide challenge.

Binding of lipopolysaccharide (LPS) to CD14 is required for its cellular effects via TLR4. A role of LPS/TLR4-mediated signaling in activated hepatic stellate cells (aHSCs), the major fibrogenic cells, in liver fibrosis has been reported. We investigated effects of LPS on carbon tetrachloride (CCl4)-induced fibrosis in CD14-knockout (KO) mice in vivo, and culture-activated HSCs in vitro. CCl4 (biweekly; 4 weeks)-treated wild type (WT) and CD14-KO mice were challenged with single LPS administration for 24 h. Liver injury, inflammation and fibrosis were determined. Culture-activated HSCs from WT or CD14-KO mice were stimulated with LPS. Parameters of fibrogenic activity (expression of collagen1a1 [Col1a1], α-smooth muscle actin [αSMA] and TGFß1) and inflammatory cytokines/chemokines were measured. CCl4 treatment caused similar liver injury and fibrosis in WT and CD14-KO mice. LPS increased liver injury and inflammation similarly in CCl4-treated WT and CD14-KO mice, but downregulated Timp1 and upregulated Mmp13. LPS elicited similar NFκB activation and inflammatory response in WT and CD14-KO aHSCs. LPS similarly downregulated Acta2 (encodes αSMA), Pdgfrb, Col1a1 and Mmp13 expression but did not affect Timp1 expression in WT and CD14-KO aHSCs. LPS did not alter Tgfb1 but increased expression of decorin (Dcn) (inhibitor of TGFß1) expression in WT and CD14-KO aHSCs. The results indicate that the effects of LPS on HSCs are CD14-independent, and CD14 is not required for hepatic fibrosis. LPS-induced down-modulation of fibrogenic markers in aHSCs is also CD14-independent.

Stellate cell in hepatic inflammation and acute injury.

The perisinusoidal hepatic stellate cells (HSCs) have been investigated extensively for their role as the major fibrogenic cells during chronic liver injury. HSCs also produce numerous cytokines, chemokines, and growth mediators, and express cell adhesion molecules constitutively and in response to stimulants such as endotoxin (lipopolysaccharide). With this property and by interacting with resident and recruited immune and inflammatory cells, HSCs regulate hepatic immune homeostasis, inflammation, and acute injury. Indeed, experiments with HSC-depleted animal models and cocultures have provided evidence for the prominent role of HSCs in the initiation and progression of inflammation and acute liver damage due to various toxic agents. Thus HSCs and/or mediators derived thereof during acute liver damage may be considered as potential therapeutic targets.

Differential recruitment of monocyte-derived macrophages in control and stellate cell-depleted mice during recurrent carbon tetrachloride-induced acute liver injury.

Liver depleted of hepatic stellate cells (HSCs) is resistant to ischemia/reperfusion-, concanavalin A-, and acetaminophen-induced acute injury. Whether HSCs regulate carbon tetrachloride (CCl4 )-induced acute liver injury is not known. CCl4 treatment damages pericentral hepatocytes that express CCl4 -metabolizing Cyp2E1 and activates HSCs. We investigated whether HSC-depletion in mice transgenic for thymidine kinase under the glial fibrillary acidic protein promoter (GFAP-TK-Tg) confers resistance to injury and inflammation due to CCl4 rechallenge. GFAP-TK-Tg or wild type (WT) mice were administered 0.16 ml/kg CCl4 (3× at 3 days intervals), then 40 µg/g/day ganciclovir for 10 days. The treatment depletes ~70%-75% HSCs from GFAP-TK-Tg but not WT mice while the liver recovers from earlier CCl4 -induced injury. Mice were then administered CCl4 , and liver injury and inflammation were determined at 24 h. HSC-depleted and HSC-sufficient mice showed similar CCl4 -induced hepatocyte necrosis and oxidative stress. However, increase in F4/80+ macrophages, but not CD68+ cells, was greater in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. Expression of tumor necrosis factor-α (TNF-α), CCL2, and CXCL1 increased similarly, whereas increase in interleukin-6 (IL6), IL1ß, and IL10 expression was higher in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. CCl4 rechallenge of HSC-sufficient mice rapidly activated HSCs causing significant fibrosis with increased expression of Col1a1, transforming growth factor ß1 (TGFß1), tissue inhibitors of metalloproteinases 1 (TIMP1); increase in TIPM1 was much lower and metalloproteinases 13 (MMP13) greater in CCl4 rechallenged HSC-depleted mice. Interestingly, hepatic recruitment of both profibrogenic (Ly6Chi ) and antifibrogenic restorative (Ly6Clo ) macrophages, and neutrophils was significantly greater in CCl4 rechallenged HSC-depleted mice. These data suggest that CCl4 directly damages hepatocytes but HSCs regulate inflammation. Rapid fibrogenesis in CCl4 rechallenged HSC-sufficient mice recovered from earlier injury indicates that even transiently activated HSCs that had reverted to the quiescent phenotype remain primed to become reactivated.

Augmenter of liver regeneration: Mitochondrial function and steatohepatitis.

Augmenter of liver regeneration (ALR), a ubiquitous fundamental life protein, is expressed more abundantly in the liver than other organs. Expression of ALR is highest in hepatocytes, which also constitutively secrete it. ALR gene transcription is regulated by NRF2, FOXA2, SP1, HNF4α, EGR-1 and AP1/AP4. ALR's FAD-linked sulfhydryl oxidase activity is essential for protein folding in the mitochondrial intermembrane space. ALR's functions also include cytochrome c reductase and protein Fe/S maturation activities. ALR depletion from hepatocytes leads to increased oxidative stress, impaired ATP synthesis and apoptosis/necrosis. Loss of ALR's functions due to homozygous mutation causes severe mitochondrial defects and congenital progressive multiorgan failure, suggesting that individuals with one functional ALR allele might be susceptible to disorders involving compromised mitochondrial function. Genetic ablation of ALR from hepatocytes induces structural and functional mitochondrial abnormalities, dysregulation of lipid homeostasis and development of steatohepatitis. High-fat diet-fed ALR-deficient mice develop non-alcoholic steatohepatitis (NASH) and fibrosis, while hepatic and serum levels of ALR are lower than normal in human NASH and NASH-cirrhosis. Thus, ALR deficiency may be a critical predisposing factor in the pathogenesis and progression of NASH.

Endotoxin-Stimulated Hepatic Stellate Cells Augment Acetaminophen-Induced Hepatocyte Injury.

Acetaminophen (APAP)-induced liver injury is influenced by inflammatory Gram-negative bacterial endotoxin [lipopolysaccharide (LPS)], mechanisms of which are not completely understood. Because LPS-stimulated perisinusoidal hepatic stellate cells (HSCs) produce cytokines that affect survival of hepatocytes, this study investigated their role in APAP-induced liver injury. Fed (nonstarved) rats were administered 5 mg/kg LPS or phosphate-buffered saline (PBS) vehicle, followed by 200 mg/kg APAP or PBS an hour later, and euthanized at 6 hours. Control rats received PBS at both time points. Both LPS and APAP caused mild hepatocyte injury (apoptosis), as assessed by histopathology, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase-3 activation. The liver injury was augmented in rats administered LPS + APAP, in association with increased nuclear translocation of interferon-regulatory factor-1 (IRF1). In vitro, APAP augmented LPS/HSC-conditioned medium-induced inhibition of DNA and protein synthesis, apoptosis, and nuclear IRF1 in hepatocytes. LPS-stimulated HSCs produced interferon-ß (IFN-ß), and LPS/HSC + APAP-induced hepatocyte apoptosis was inhibited by anti-IFN-ß antibody. Finally, HSC-depleted mice produced significantly lower IFN-ß and tumor necrosis factor-α, exhibited less oxidative stress, and were protected from excessive injury due to high APAP dose (600 mg/kg), as well as LPS (5 mg/kg overnight) followed by APAP. In co-culture with or without LPS, HSCs increased expression of proinflammatory cytokines by Kupffer cells. These results suggest that HSCs play a critical role in APAP-induced liver injury without or with LPS preconditioning, and it involves INF-ß-IRF1 signaling.

MRI Measures of Murine Liver Fibrosis.

BACKGROUND: An imaging method that allows quantitative fibrosis estimates is needed to facilitate the diagnosis of chronic liver disease. Amide proton transfer (APT) and tissue sodium concentration (TSC) estimates could meet this need. HYPOTHESIS: APT and TSC estimates correlate with fibrosis in a mouse model of chronic liver disease. STUDY TYPE: Prospective. PHANTOMS/ANIMAL MODEL: Male C57Bl/6 mice given CCl4 or vehicle (N = 8 each) twice weekly for 16 weeks. FIELD STRENGTH/SEQUENCE: Liver T1 (Look-Locker gradient recalled echo [GRE] sequence), T2 (multiecho spin echo sequence), T1rho (fast spin echo sequence with 500 Hz spin locking pulse), and APT (GRE sequence with off-resonance pulses) data were acquired at 7 T at 12 and 16 weeks. Liver sodium data (multiple echo GRE sequence) were acquired at 12 weeks at 9.4 T. ASSESSMENT: Liver proton T1 , T2 , T1rho , APT, sodium T2 *, and TSC were calculated. Histological measures included Sirius Red, hematoxylin and eosin, liver hydroxyproline content, and serum alanine transaminase (ALT). STATISTICAL TESTS: Welch's two-sided t-test was used to test for differences between control and CCl4 -treated groups for serum ALT, hydroxyproline, Sirius Red staining, T1 , T2 , T1rho , APT, TSC, and sodium T2 *. Pearson's correlations between liver T1 , APT, TSC, or sodium T2 * with Sirius Red staining and hydroxyproline levels were calculated. RESULTS: APT was significantly different (P < 0.05) between groups in the left liver lobe at 16 weeks (CCl4 : 8.0% ± 1.2%, controls: 6.2% ± 1.0%), as were average liver TSC at 12 weeks (CCl4 : 38 mM ± 5 mM, controls: 27 mM ± 2 mM), and average sodium liver T2 * at 12 weeks (CCl4 : 10 msec ± 1.0 msec, controls: 12 msec ± 1.9 msec). APT, TSC, and sodium T2 * correlated significantly (P < 0.05) with Sirius Red staining and hydroxyproline levels. DATA CONCLUSION: Liver TSC and APT significantly correlated with histopathologic markers of fibrosis in this mouse model. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 3.

Pro- and Anti-fibrogenic Functions of Gram-Negative Bacterial Lipopolysaccharide in the Liver.

Extensive research performed over several decades has identified cells participating in the initiation and progression of fibrosis, and the numerous underlying inter- and intra-cellular signaling pathways. However, liver fibrosis continues to be a major clinical challenge as the precise targets of treatment are still elusive. Activation of physiologically quiescent perisinusoidal hepatic stellate cells (HSCs) to a myofibroblastic proliferating, contractile and fibrogenic phenotype is a critical event in the pathogenesis of chronic liver disease. Thus, elucidation of the mechanisms of the reversal to quiescence or inhibition of activated HSCs, and/or their elimination via apoptosis has been the focus of intense investigation. Lipopolysaccharide (LPS), a gut-resident Gram-negative bacterial endotoxin, is a powerful pro-inflammatory molecule implicated in hepatic injury, inflammation and fibrosis. In both acute and chronic liver injury, portal venous levels of LPS are elevated due to increased intestinal permeability. LPS, via CD14 and Toll-like receptor 4 (TLR4) and its adapter molecules, stimulates macrophages, neutrophils and several other cell types to produce inflammatory mediators as well as factors that can activate HSCs and stimulate their fibrogenic activity. LPS also stimulates synthesis of pro- and anti-inflammatory cytokines/chemokines, growth mediators and molecules of immune regulation by HSCs. However, LPS was found to arrest proliferation of activated HSCs and to convert them into non-fibrogenic phenotype. Interestingly, LPS can elicit responses in HSCs independent of CD14 and TLR4. Identifying and/or developing non-inflammatory but anti-fibrogenic mimetics of LPS could be relevant for treating liver fibrosis.

Hepatic Deficiency of Augmenter of Liver Regeneration Predisposes to Nonalcoholic Steatohepatitis and Fibrosis.

BACKGROUND AND AIMS: The augmenter of liver regeneration (ALR) protein is critical for lipid homeostasis and mitochondrial function. We investigated high-fat/high-carbohydrate (HF/HC) diet-induced nonalcoholic fatty liver disease (NAFLD) in wild-type (WT), hepatocyte-specific ALR-knockout (ALR-H-KO), and ALR-heterozygous (ALR-H-HET) mice. ALR was measured in serum of human nonalcoholic steatohepatitis (NASH) and NASH-induced cirrhosis (serum and liver). APPROACH AND RESULTS: HF/HC feeding decreased ALR expression in all groups of mice. The otherwise normal ALR-H-HET mice gained more weight and steatosis than WT mice when challenged metabolically with the HF/HC diet; ALR-H-KO mice gained the least weight and had the least steatosis. These findings were consistent with correspondingly increased triglycerides and cholesterol and altered expression of carnitine palmitoyltransferase 1a, sterol regulatory element-binding protein, acetyl coenzyme A carboxylase, and fatty acid synthase. All HF/HC-fed mice developed insulin resistance, the magnitude being lower in ALR-H-KO mice. HF/HC-fed ALR-H-HET mice were more resistant to glucose challenge than WT or ALR-H-KO mice. The frequency of tumor necrosis factor alpha-producing, interleukin 6 (IL6)-producing, and IL17-producing cells was greater in ALR-H-KO than ALR-H-HET and lowest in WT mice. HF/HC feeding did not increase their number in ALR-H-KO mice, and the increase in ALR-H-HET was greater than that in WT mice except for IL17 cells. Cluster of differentiation 25-positive (CD25+ ) forkhead box P3-positive CD4+ regulatory T-cell frequency was lower in ALR-H-HET than WT mice and further reduced in ALR-H-KO mice; HF/HC reduced regulatory T-cell frequency only in WT mice. HF/HC-fed ALR-H-HET, but not WT, mice developed fibrosis; and ALR-H-KO mice progressed to cirrhosis. White adipose tissue of HF/HC-fed ALR-deficient mice developed strong inflammation, indicating bidirectional interactions with the liver. Hepatic and serum ALR levels were significantly reduced in patients with NASH-cirrhosis. Serum ALR was also significantly lower in patients with NASH. CONCLUSIONS: Hepatic ALR deficiency may be a critical predisposing factor for aggressive NAFLD progression.

Lipopolysaccharide Reverses Hepatic Stellate Cell Activation Through Modulation of cMyb, Small Mothers Against Decapentaplegic, and CCAAT/Enhancer-Binding Protein C/EBP Transcription Factors.

BACKGROUND AND AIMS: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFßR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. APPROACH AND RESULTS: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFßR, transforming growth factor beta receptor 1 (TGFßR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFßR, αSMA, TGFßR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. CONCLUSIONS: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.

Yes-Associated Protein in Kupffer Cells Enhances the Production of Proinflammatory Cytokines and Promotes the Development of Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Yes-associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non-neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of proinflammatory cytokines and promotes the development of nonalcoholic steatohepatitis (NASH). Our data show that the expression of YAP is significantly increased in KCs of wild-type mice fed a high-fat diet (HFD). APPROACH AND RESULTS: We generated mice with macrophage/monocyte-specific deletion of YAP (YAPϕKO ) or Toll-like receptor 4 (TLR4; TLR4ϕKO ), and animals were fed an HFD or treated with lipopolysaccharide (LPS). Our data showed that YAPϕKO mice fed an HFD exhibited lower serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced accumulation of YAP in KCs in vitro and in mice, which was prevented by macrophage/monocyte-specific deletion of TLR4 (TLR4ϕKO ). LPS transcriptionally activates YAP through activator protein 1 in macrophages/KCs. LPS-induced YAP further enhances expression of proinflammatory cytokines (including monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin 6) through YAP association with the TEA domain-binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of proinflammatory cytokines. Treatment of HFD-fed mice with verteporfin inhibited KC activation, reduced liver inflammation, and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that level of YAP in human liver tissues is positively correlated with expression of proinflammatory cytokines. CONCLUSIONS: This study describes an important role of YAP in KCs for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent an effective therapeutic strategy for NASH treatment.

The Forkhead box F1 transcription factor inhibits collagen deposition and accumulation of myofibroblasts during liver fibrosis.

Hepatic fibrosis is the common end stage to a variety of chronic liver injuries and is characterized by an excessive deposition of extracellular matrix (ECM), which disrupts the liver architecture and impairs liver function. The fibrous lesions are produced by myofibroblasts, which differentiate from hepatic stellate cells (HSC). The myofibroblast's transcriptional networks remain poorly characterized. Previous studies have shown that the Forkhead box F1 (FOXF1) transcription factor is expressed in HSCs and stimulates their activation during acute liver injury; however, the role of FOXF1 in the progression of hepatic fibrosis is unknown. In the present study, we generated αSMACreER;Foxf1fl/fl mice to conditionally inactivate Foxf1 in myofibroblasts during carbon tetrachloride-mediated liver fibrosis. Foxf1 deletion increased collagen depositions and disrupted liver architecture. Timp2 expression was significantly increased in Foxf1-deficient mice while MMP9 activity was reduced. RNA sequencing of purified liver myofibroblasts demonstrated that FOXF1 inhibits expression of pro-fibrotic genes, Col1α2, Col5α2, and Mmp2 in fibrotic livers and binds to active repressors located in promotors and introns of these genes. Overexpression of FOXF1 inhibits Col1a2, Col5a2, and MMP2 in primary murine HSCs in vitro Altogether, FOXF1 prevents aberrant ECM depositions during hepatic fibrosis by repressing pro-fibrotic gene transcription in myofibroblasts and HSCs.

Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.

Levels of augmenter of liver regeneration (ALR), a multifunctional protein, are reduced in steatohepatitis. ALR depletion from ALR flox/flox/Alb-Cre [ALR-L-knockout (KO)] mouse causes robust steatosis and apoptosis of hepatocytes, and pericellular fibrosis between 1 and 2 wk postbirth. Steatosis regresses by 4 wk upon reappearance of ALR-expressing hepatocytes. We investigated mechanisms of ALR depletion-induced steatosis. ALR-L-KO mice (1-, 2-, and 4 wk old) and Adeno-Cre-transfected ALR flox/flox hepatocytes were used for in vivo and in vitro studies. ALR depletion from hepatocytes in vivo downregulated peroxisome proliferator-activated receptor (PPAR)-α, carnitine palmitoyl transferase I (CPT1)a, peroxisomal membrane protein 70 (PMP70) (modest down-regulation), and acyl-CoA oxidase 1 (ACOX1). The markedly up-regulated (20X) novel microRNA-540 (miR-540) was identified to target PPARα, PMP70, ACOX1, and CPT1a. ALR depletion from primary hepatocytes increased oxidative stress, miR-540 expression, and steatosis and down-regulated PPARα, ACOX1, PMP70, and CPT1a expression. Anti-miR-540 mitigated ALR depletion-induced steatosis and prevented loss of PPARα, ACOX1, PMP70, and CPT1a expression. Antioxidant N-acetylcysteine and recombinant ALR (rALR) both inhibited ALR depletion-induced miR-540 expression and lipid accumulation in hepatocytes. Finally, treatment of ALR-L-KO mice with rALR between 1 and 2 wk prevented miR-540 expression, and arrested steatosis and fibrosis. We conclude that ALR deficiency-mediated oxidative stress induces generation of miR-540, which promotes steatosis by dysregulating peroxisomal and mitochondrial lipid homeostasis.-Kumar, S., Rani, R., Karns, R., Gandhi, C. R. Augmenter of liver regeneration protein deficiency promotes hepatic steatosis by inducing oxidative stress and microRNA-540 expression.

Mechanisms of concanavalin A-induced cytokine synthesis by hepatic stellate cells: Distinct roles of interferon regulatory factor-1 in liver injury.

Mice depleted of hepatic stellate cells (HSCs) are protected from concanavalin A (ConA)-induced liver injury that is mediated by the activation of interferon regulatory factor 1 (IRF1). The aim of this study was to determine the mechanisms of ConA-mediated signaling and synthesis/release of mediators by HSCs that damage hepatocytes. Primary cultures of wildtype (WT) and IRF1-knockout (KO) HSCs and hepatocytes were used, and ConA-induced liver damage in interferon (IFN)αß receptor-deficient (IFNαßR-KO) mice was determined. Specific binding of ConA to HSCs induced rapid activation of JAK2 and STAT1. ConA-induced expression of IRF1, IFNß, tumor necrosis factor α, and CXCL1 was abrogated by selective inhibition of JAK2 and STAT1. Despite activating JAK2/STAT1, ConA failed to stimulate expression of inflammatory cytokines in HSCs from IRF1-KO mice. ConA-conditioned WT-HSC medium caused activation of JNK and caspase 3, and apoptosis of hepatocytes from WT but not from IRF1-KO or IFNαßR-KO mice. Conversely, ConA-conditioned medium of IRF1-KO HSCs failed to cause apoptosis of WT or IRF1-KO hepatocytes. IFNαßR-KO mice were protected from ConA-induced liver damage, and ConA-induced hepatic expression of IRF1 and pro-inflammatory cytokines and chemokines, and infiltration of neutrophils were significantly lower in IFNαßR-KO than in WT mice. These results demonstrate distinct roles of IRF1 in hepatic inflammation (HSCs) and injury (hepatocytes) and can be an important target for intervention in acute liver injury.

Stellate Cells Orchestrate Concanavalin A-Induced Acute Liver Damage.

Concanavalin A (ConA) causes immune cell-mediated liver damage, but the contribution of resident nonparenchymal cells (NPCs) is also evident. Hepatic stellate cells (HSCs) induce hepatic inflammation and immunological reactions; we therefore investigated their role in ConA-induced liver injury. ConA was administered i.v. to control or HSC-depleted mice; hepatic histopathology and cytokines/chemokines were determined after 6 hours. In vitro, effects of ConA-conditioned HSC medium on hepatocytes were determined. ConA induced inflammation, sinusoidal congestion, and extensive midzonal hepatocyte death in control mice, which were strongly minimized in HSC-depleted mice. CD4 and natural killer T cells and neutrophils were markedly reduced in ConA-treated HSC-depleted mice compared with control mice. The increase in cytokines/chemokines of hepatic injury was much higher in ConA-treated control mice than in HSC-depleted mice. ConA-treated HSCs showed increased expression of interferon-ß, tumor necrosis factor-α, and CXCL1, induced oxidative stress in hepatocytes, and caused hepatocyte apoptosis. ConA induced nuclear translocation of interferon-regulatory factor-1 (IRF1) in hepatocytes in vivo, and ConA/HSC induced a similar effect in cultured hepatocytes. IRF1-knockout mice were resistant to ConA-induced liver damage, and anti-interferon ß antibody mitigated ConA/HSC-induced injury. In HSC-NPC co-culture, ConA-induced expression of inflammatory cytokines/chemokines was significantly augmented compared with NPCs alone. HSCs play an essential role in ConA-induced liver injury directly via the interferon-ß/IRF1 axis, and by modulating properties of NPCs.

Toll-Like Receptor 4-Independent Carbon Tetrachloride-Induced Fibrosis and Lipopolysaccharide-Induced Acute Liver Injury in Mice: Role of Hepatic Stellate Cells.

Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is implicated in acute and chronic liver injury; its effects are mediated predominantly via the membrane receptor Toll-like receptor 4 (TLR4). However, TLR4-independent effects of LPS may play important role in hepatic pathophysiology. We investigated carbon tetrachloride (CCl4)-induced fibrosis and LPS-induced acute liver injury in wild-type (WT) and B6.B10ScN-Tlr4lps-del/JthJ [TLR4-knockout (KO)] mice. Effects of LPS on fibrogenic hepatic stellate cells (HSCs) from WT and TLR4-KO mice were assessed in vitro. CCl4 produced similar fibrosis and necroinflammation and increased the mRNA and protein expression of cytokines and chemokines in WT and TLR4-KO mice. However, circulating LPS concentration did not increase in CCl4-treated mice. Interestingly, LPS down-modulated α-smooth muscle actin (activated HSC marker) and collagen 1 in both WT and TLR4-KO HSCs. LPS induced similar activation of NF-κB, and stimulated the expression of cytokines and chemokines in WT and TLR4-KO HSCs. Finally, LPS caused similar inflammation and injury in previously untreated WT and TLR4-KO mice. The results provide evidence of the TLR4/LPS-independent mechanisms of liver fibrosis and also indicate that TLR4 is not entirely critical to LPS-induced acute liver injury. The results further indicate that LPS signaling in activated HSCs might be a mechanism of limiting liver fibrosis.

15-hydroxyprostaglandin dehydrogenase (15-PGDH) prevents lipopolysaccharide (LPS)-induced acute liver injury.

The NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of prostaglandin E2 (PGE2), converting the pro-inflammatory PGE2 to the anti-inflammatory 15-keto-PGE2 (an endogenous ligand for peroxisome proliferator-activated receptor-gamma [PPAR-γ]). To evaluate the significance of 15-PGDH/15-keto-PGE2 cascade in liver inflammation and tissue injury, we generated transgenic mice with targeted expression of 15-PGDH in the liver (15-PGDH Tg) and the animals were subjected to lipopolysaccharide (LPS)/Galactosamine (GalN)-induced acute liver inflammation and injury. Compared to the wild type mice, the 15-PGDH Tg mice showed lower levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), less liver tissue damage, less hepatic apoptosis/necrosis, less macrophage activation, and lower inflammatory cytokine production. In cultured Kupffer cells, treatment with 15-keto-PGE2 or the conditioned medium (CM) from 15-PGDH Tg hepatocyes inhibited LPS-induced cytokine production, in vitro. Both 15-keto-PGE2 and the CM from15-PGDH Tg hepatocyes also up-regulated the expression of PPAR-γ downstream genes in Kupffer cells. In cultured hepatocytes, 15-keto-PGE2 treatment or 15-PGDH overexpression did not influence TNF-α-induced hepatocyte apoptosis. These findings suggest that 15-PGDH protects against LPS/GalN-induced liver injury and the effect is mediated via 15-keto-PGE2, which activates PPAR-γ in Kupffer cells and thus inhibits their ability to produce inflammatory cytokines. Accordingly, we observed that the PPAR-γ antagonist, GW9662, reversed the effect of 15-keto-PGE2 in Kupffer cell in vitro and restored the susceptibility of 15-PGDH Tg mice to LPS/GalN-induced acute liver injury in vivo. Collectively, our findings suggest that 15-PGDH-derived 15-keto-PGE2 from hepatocytes is able to activate PPAR-γ and inhibit inflammatory cytokine production in Kupffer cells and that this paracrine mechanism negatively regulates LPS-induced necro-inflammatory response in the liver. Therefore, induction of 15-PGDH expression or utilization of 15-keto-PGE2 analogue may have therapeutic benefits for the treatment of endotoxin-associated liver inflammation/injury.